The project was initiated to study the regulation of growth and differentiation of normal and transformed cells using the hepatocyte model in combination with quantitative two-dimensional electrophoresis. Cultured hepatocytes were treated with non-toxic concentrations of the hepatocarcinogen N-hydroxy-2-acetylamino-fluorene (N-OH-AAF) in order to obtain only "initiated" cell populations. Using these cells we have begun studies concerning: (1) early changes in gene expression as measured by quantitative two-dimensional gel electrophoresis of total cellular proteins; (2) the in vitro activities of sulfotransferase, deacetylase and acyltransferase, enzymes implicated in the in vivo activation of N-OH-AAF to its ultimate carcinogenic form; (3) identification and quantitation using P-32 postlabeling analysis of cellular carcinogen bound (fluorene) DNA adducts formed during initiation. Treatment of cultured hepatocytes with N-OH-AAF (0.1 Mug/ml) resulted in a transient inhibition of protein synthesis as measured by incorporation of C-14 amino acids into cellular proteins which returned to control levels 72 hours after exposure. Treated cells, however, showed no morphological differences when compared to untreated hepatocytes. Two-dimensional electrophoretic analysis of total cellular proteins from treated and untreated cells revealed few (6-8) qualitative protein changes between the groups. In contrast 10-15% of the 600-800 readily detectable proteins were undergoing quantitative changes of at least 50% following treatment with N-OH-AAF. In order to examine the relationship between the covalent binding of carcinogenic (aromatic amines) to cellular DNA with gene expression in early initiated cells, we have synthesized the major known or proposed deoxyguanosine and deoxyadenosin DNA adducts of N-OH-AAF. In addition the respective 3-mono-phosphate and 3 feet, 5 feet -diphosphate adducts have also been prepared and we are currently developing a high pressure liquid chromatographic procedure for the separation and quantitation of the in vitro formed cellular DNA adducts.